Mark L. Witten

Temporal Variability of Tungsten and Cobalt in Fallon, Nevada

Background Since 1997, Fallon, Nevada, has experienced a cluster of childhood leukemia that has been declared “one of the most unique clusters of childhood cancer ever reported.” Multiple environmental studies have shown airborne tungsten and cobalt to be elevated within Fallon, but the question remains: Have these metals changed through time in correspondence with the onset of the leukemia cluster? Methods We used dendrochemistry, the study of element concentrations through time in tree rings, in Fallon to assess temporal variability of airborne tungsten and cobalt since the late 1980s. The techniques used in Fallon were also tested in a different town (Sweet Home, OR) that has airborne tungsten from a known source. Results The Sweet Home test case confirms the accuracy of dendrochemistry for showing temporal variability of environmental tungsten. Given that dendrochemistry works for tungsten, tree-ring chemistry shows that tungsten increased in Fallon relative to nearby comparison towns beginning by the mid-1990s, slightly before the onset of the cluster, and cobalt has been high throughout the last ~ 15 years. Other metals do not show trends through time in Fallon. Discussion Results in Fallon suggest a temporal correspondence between the onset of excessive childhood leukemia and elevated levels of tungsten and cobalt. Although environmental data alone cannot directly link childhood leukemia with exposure to metals, research by others has shown that combined exposure to tungsten and cobalt can be carcinogenic to humans. Conclusion Continued biomedical research is warranted to directly test for linkage between childhood leukemia and tungsten and cobalt.

Strategies for evaluating the environment–public health interaction of long-term latency disease: The quandary of the inconclusive case–control study

Environmental links to disease are difficult to uncover because environmental exposures are variable in time and space, contaminants occur in complex mixtures, and many diseases have a long time delay between exposure and onset. Furthermore, individuals in a population have different activity patterns (e.g., hobbies, jobs, and interests), and different genetic susceptibilities to disease. As such, there are many potential confounding factors to obscure the reasons that one individual gets sick and another remains healthy. An important method for deducing environmental associations with disease outbreak is the retrospective case-control study wherein the affected and control subject cohorts are studied to see what is different about their previous exposure history. Despite success with infectious diseases (e.g., food poisoning, and flu), case-control studies of cancer clusters rarely have an unambiguous outcome. This is attributed to the complexity of disease progression and the long-term latency between exposure and disease onset. In this article, we consider strategies for investigating cancer clusters and make some observations for improving statistical power through broader non-parametric approaches wherein sub-populations (i.e., whole towns), rather than individuals, are treated as the cases and controls, and the associated cancer rates are treated as the dependent variable. We subsequently present some ecological data for tungsten and cobalt from studies by University of Arizona researchers who document elevated levels of tungsten and cobalt in Fallon, NV. These results serve as candidates for future hybrid ecologic case-control investigations of childhood leukemia clusters.

Dose-dependent transcriptome changes by metal ores on a human acute lymphoblastic leukemia cell line

The increased morbidity of childhood leukemia in Fallon, Nevada and Sierra Vista, Arizona has prompted great health concern. The main characteristic that these two towns share is the environmental pollution attributed to metal ore from abandoned mining operations. Consequently, we have investigated the transcriptome effects of metal ores from these endemic areas using a human T-cell acute lymphoblastic leukemia cell line (T-ALL). Metal ore from Fallon significantly increased cell growth after 24, 48 and 72 h of incubation at 1.5 mg/mL concentration, as measured by trypan-blue. Sierra Vista ore significantly increased cell growth with 0.15 and 1.5 mg/mL following 72 h of incubation. From human cDNA microarray, results indicate that in total, eight genes, mostly metallothionein (MT) genes, were up-regulated and 10 genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 mg/mL of metal ores at 72 h, in comparison with untreated cells. Twenty-eight metals of both ores were quantified and their presence may be associated with the cell growth rate and dose-dependent activation of transcriptomes in immature T-cells.

Exposure to sodium tungstate and Respiratory Syncytial Virus results in hematological/immunological disease in C57BL/6J mice

The etiology of childhood leukemia is not known. Strong evidence indicates that precursor B-cell Acute Lymphoblastic Leukemia (Pre-B ALL) is a genetic disease originating in utero. Environmental exposures in two concurrent, childhood leukemia clusters have been profiled and compared with geographically similar control communities. The unique exposures, shared in common by the leukemia clusters, have been modeled in C57BL/6 mice utilizing prenatal exposures. This previous investigation has suggested in utero exposure to sodium tungstate (Na2WO4) may result in hematological/immunological disease through genes associated with viral defense. The working hypothesis is (1) in addition to spontaneously and/or chemically generated genetic lesions forming pre-leukemic clones, in utero exposure to Na2WO4 increases genetic susceptibility to viral influence(s); (2) postnatal exposure to a virus possessing the 1FXXKXFXXA/V9 peptide motif will cause an unnatural immune response encouraging proliferation in the B-cell precursor compartment. This study reports the results of exposing C57BL/6J mice to Na2WO4 in utero via water (15 ppm, ad libetum) and inhalation (mean concentration PM5 3.33 mg/m3) and to Respiratory Syncytial Virus (RSV) within 2 weeks of weaning. Inoculation of C57BL/6J mice with RSV was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with Na2WO4-exposure, significant splenomegaly resulted (p=0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only, respectively) in addition to other hematological pathologies which were not significant. Exposure to Na2WO4 and RSV resulted in hematological/immunological disease, the nature of which is currently inconclusive. Further research is needed to characterize this potential leukemia mouse model.

Morphological and Chemical Characteristics of Airborne Tungsten Particles of Fallon, Nevada

Morphological and chemical characteristics were determined for airborne tungsten particles in Fallon, Nevada, a town that is distinguishable environmentally by elevated airborne tungsten and cobalt. From samples of airborne dust collected previously at six different places in Fallon, tungsten-rich dust particles were isolated and analyzed with automated electron microprobe and wavelength-dispersive spectrometry. Representative W particles were further analyzed using transmission electron microscopy. Morphologically, Fallon W particles are angular and small, with minimum and maximum sizes of < or = 1 microm and 5.9 microm in diameter, respectively. The number and size of tungsten-rich particles decrease in Fallon with distance from a hard-metal facility located near the center of town. Chemically, Fallon airborne W particles include mixtures of tungsten with cobalt plus other metals such as chromium, iron, and copper. No W-rich particles were identifiable as CaWO4 (scheelite) or MnWO4 (huebnerite). From d-spacings, Fallon particles are most consistent with identification as tungsten carbide. Based on these multiple lines of evidence, airborne W particles in Fallon are anthropogenic in origin, not natural. The hard-metal facility in Fallon processes finely powdered W and W-Co, and further investigation using tracer particles is recommended to definitively identify the source of Fallons airborne tungsten.

Spatial patterns of tungsten and cobalt on leaf surfaces of trees in Fallon , Nevada

Spatial patterns of airborne tungsten and cobalt are described from leaf-surface chemistry of trees in Fallon, Nevada, where a cluster of childhood leukemia has been ongoing since 1997. In earlier research, airborne tungsten and cobalt have been shown to be elevated in total suspended particulates, surface dust, and lichens from Fallon. To update data on the spatial patterns of airborne tungsten and cobalt in Fallon, leaves were collected in October 2007 from trees growing throughout Fallon. Collected leaves were measured for metals accumulated onto their surfaces. On Fallon leaf surfaces, tungsten and cobalt show maxima of 17 ppm and 6 ppm, respectively, near the center of town, north of Highway 50 and west of Highway 95. These two peaks overlap spatially, and given the dense and widespread pattern of collection, the source area of these two airborne metals can be pinpointed to the vicinity of a hard-metal industry located north of Highway 50 and west of Highway 95. Fallon is distinctive in west central Nevada for its elevated airborne tungsten and cobalt particulates, and given its cluster of childhood leukemia cases, it stands to reason that additional biomedical research is in order to test directly the leukogenicity of combined airborne tungsten and cobalt particulates.

Laser Trimming Tree-Ring Cores for Dendrochemistry of Metals

This article discusses the application of laser to trim the outer surface from tree-ring increment cores in preparation for dendrochemistry of certain metals. A source of contamination specific to dendrochemistry of metals is metal constituents, such as iron, tungsten, chromium, nickel, and cobalt, coming off tools used to collect and process cores and adhering to the sample surface. One method to eliminate this contamination is to trim off the outer surface of cores using laser. To test this application of laser, three tree-ring increment cores were collected from each of three trees. For each tree, one core was trimmed using a CO2 laser, one core was trimmed using a stainless steel razor blade, and one core was left untrimmed. The resultant cores were measured for metals using acid dissolution inductively coupled plasma mass spectroscopy. Trimmed cores had on average one-third the content of iron, tungsten, and chromium than that of untrimmed cores. Laser-trimmed cores had less of these metals than razor-trimmed cores. Razor-trimmed cores also had measurable nickel, but laser-trimmed cores had no nickel. Laser trimming is an ideal solution to potential contamination of cores with metals from increment borers without imparting other contamination from tools such as razor blades.

Comparison of Size and Geography of Airborne Tungsten Particles in Fallon, Nevada, and Sweet Home, Oregon, with Implications for Public Health

To improve understanding of possible connections between airborne tungsten and public health, size and geography of airborne tungsten particles collected in Fallon, Nevada, and Sweet Home, Oregon, were compared. Both towns have industrial tungsten facilities, but only Fallon has experienced a cluster of childhood leukemia. Fallon and Sweet Home are similar to one another by their particles of airborne tungsten being generally small in size. Meteorologically, much, if not most, of residential Fallon is downwind of its hard metal facility for at least some fraction of time at the annual scale, whereas little of residential Sweet Home is downwind of its tungsten facility. Geographically, most Fallon residents potentially spend time daily within an environment containing elevated levels of airborne tungsten. In contrast, few Sweet Home residents potentially spend time daily within an airborne environment with elevated levels of airborne tungsten. Although it cannot be concluded from environmental data alone that elevated airborne tungsten causes childhood leukemia, the lack of excessive cancer in Sweet Home cannot logically be used to dismiss the possibility of airborne tungsten as a factor in the cluster of childhood leukemia in Fallon. Detailed modeling of all variables affecting airborne loadings of heavy metals would be needed to legitimately compare human exposures to airborne tungsten in Fallon and Sweet Home.

Additional analysis of dendrochemical data of Fallon, Nevada

Previously reported dendrochemical data showed temporal variability in concentration of tungsten (W) and cobalt (Co) in tree rings of Fallon, Nevada, US. Criticism of this work questioned the use of the Mann-Whitney test for determining change in element concentrations. Here, we demonstrate that Mann-Whitney is appropriate for comparing background element concentrations to possibly elevated concentrations in environmental media. Given that Mann-Whitney tests for differences in shapes of distributions, inter-tree variability (e.g., coefficient of median variation) was calculated for each measured element across trees within subsites and time periods. For W and Co, the metals of highest interest in Fallon, inter-tree variability was always higher within versus outside of Fallon. For calibration purposes, this entire analysis was repeated at a different town, Sweet Home, Oregon, which has a known tungsten-powder facility, and inter-tree variability of W in tree rings confirmed the establishment date of that facility. Mann-Whitney testing of simulated data also confirmed its appropriateness for analysis of data affected by point-source contamination. This research adds important new dimensions to dendrochemistry of point-source contamination by adding analysis of inter-tree variability to analysis of central tendency. Fallon remains distinctive by a temporal increase in W beginning by the mid 1990s and by elevated Co since at least the early 1990s, as well as by high inter-tree variability for W and Co relative to comparison towns.

Tungsten and cobalt: Sheppard et al. Respond

First and foremost, our data from Nevada (Sheppard et al. 2007c) should not be quantitatively compared with that from Oregon. We did not make such a comparison in our article; to reinforce separation of these two studies, we described the results in separate subsections and presented data in separate figures. Our Oregon study was an independent test of dendrochemistry for establishing temporal patterns of environmental tungsten in a town with known emission of airborne tungsten. Tungsten emission in Sweet Home, Oregon, began in November 2000, and tree-ring tungsten in cottonwoods near the emission source increased at that time relative to comparison towns in central Oregon. This test demonstrated that dendrochemistry accurately depicts tungsten availability, at least when using cottonwoods. Therefore, dendrochemistry, especially when using cottonwood trees, can be trusted to accurately depict tungsten availability in Fallon, Nevada, where timing of emission of tungsten particles is not known with certainty. n nThe comparison of real interest was between Fallon and other towns of west-central Nevada. Tree-ring tungsten in Fallon was not significantly different from that of other towns of west central Nevada during the tree-ring period centered on 1991 [Figure 4 in our article (Sheppard et al. 2007c)], before the onset of the leukemia cluster. During the tree-ring period centered on 1995, corresponding to just before the onset of the leukemia cluster, Fallon tree-ring tungsten began trending upward and was significantly higher than Nevada comparison towns. During the following two time periods, overlapping temporally with the childhood leukemia cluster, Fallon tree-ring tungsten continued trending upward and remained higher than Nevada comparison towns, with significance levels at or near p = 0.05 (we provided p-values in place of error bars), thus indicating the temporal correspondence between elevated tungsten in Fallon and the childhood leukemia cluster. n nThe Centers for Disease Control and Prevention (CDC) conclusion that tungsten is not mathematically associated with the leukemia cases of Fallon (CDC 2003) is based on case-comparison testing within Fallon. This does not rule out that an underlying association actually exists but is not detectable by the case-comparison technique. Granted, no relation was reported between leukemia and tungsten exposure, but exposure to tungsten was found to be community-wide, with levels being high both in case children and families and in comparison children and families (CDC 2003). In other words, there was little to no variability in exposure at the community scale (i.e., most everyone in Fallon has been exposed) but high variability in onset of disease (i.e., some people got leukemia but others have not). When variability of an exposure is low relative to individual susceptibility to a disease, genetic studies are needed to identify gene polymorphisms that might make sick children more susceptible to effects of the exposure (Steinberg et al. 2007). n nOur environmental research in Fallon has followed an ecologic approach with the philosophy that greater variability in exposure between different towns is more important than the minor variability in exposure within communities (Sheppard et al. 2007a). The entire town of Fallon has been compared environmentally with other towns of west-central Nevada. Multiple environmental indicators have been used, such as outdoor airborne particulates (Sheppard et al. 2006), lichens (Sheppard et al. 2007d), surface dust (Sheppard et al. 2007b), and tree rings (Sheppard et al. 2007c). These indicators incorporate environmental conditions differently from one another, yet they have corroborated one another in showing that airborne tungsten is elevated in Fallon relative to other towns of west-central Nevada or the surrounding desert. Additionally, airborne tungsten particles in Fallon have been identified as anthropogenic in origin, and not natural (Sheppard et al. 2007e). n nEven with this preponderance of evidence showing spatial and temporal patterns of airborne tungsten in Fallon, we still have not concluded in any of our reports on Fallon that exposure to tungsten causes leukemia. Quite the opposite: We have acknowledged that environmental data alone cannot lead to such a conclusion and that direct biomedical testing is needed to establish a causal linkage between tungsten and leukemia. n nYears ago in an article on disease cluster research, Shimkin (1965) stated that cooperation of industrial management is needed to identify and reduce environmental carcinogens. This comment still rings true today. Kennametal Inc. (Fallon, NV) claims that it supports research in Fallon aimed at understanding the childhood leukemia cluster there (Goodale 2005), but its support is apparently selective. We hereby encourage Kennametal to engage in reasonable dialogue about research in Fallon related to the childhood leukemia cluster.